Objective To construct a lentivirus-based expression plasmid and gene knockout plasmid of human interleukin(IL)-26 so as to lay a foundation of studying the function of IL-26 gene in cell signaling pathway and autophagy.Methods IL-26 gene sequence was amplified from human peripheral blood mononuclear cells by RT-PCR and cloned into pCDH-CMVMCS-EF1-copGFP eukaryotic expression vector to construct overexpression plasmid;Four knockout targets,Exon1sgRNA1,Exon1sgRNA2,Exon3sgRNA1 and Exon3sgRNA2,were designed based on the exon sequence of IL-26,and constructed into lentiCRISPRv2 vector by CRISPR/Cas9 technology to construct gene knockout plasmid. The overexpression plasmid and gene knockout plasmid were transiently transfected into HEK293T cells respectively,and the expression of IL-26 was verified by RT-qPCR and Western blot. In addition,amino acid sequence analysis,structure prediction and subcellular localization observation of IL-26 were performed.Results The results of restriction digestion,sequencing and bioinformatics analysis showed that IL-26 was 516 bp in length,encoding 171 amino acids. The IL-26 mRNA level and protein level of HEK293T cells transfected with IL-26 overexpression plasmid increased by 656. 789 times and 1. 978 times respectively with significant differences as compared with the normal control group(t = 17. 976 and 7. 859,P < 0. 000 1 and < 0. 001,respectively). With the transfection of 4 knockout targets Exon1sgRNA1,Exon1sgRNA2,Exon3sgRNA1 and Exon3sg-RNA2 into HEK293T cells,the expression of IL-26 decreased by 0. 930,0. 980,0. 523 3 and 0. 316 9 times,respectively,among which Exon3sgRNA2 significantly down-regulated the expression of IL-26(t = 7. 440,P < 0. 001). IL-26protein showed signal peptide structure and certain transmembrane function in the first 22 amino acids,which existed in cytoplasm.Conclusion IL-26overexpression and gene knockout plasmids were successfully constructed,which laid a foundation of the follow-up study of the function of IL-26.

Objective To evaluate the significance of combined detection of leptin (LEP),tumor necrosis factor-α (TNF-α),carcino-embryonic antigen (CEA),C-reactive protein (CRP) and interleukin-6 (IL-6) in both serum and pleural effusion in differential diagnosis of tuberculosis and malignant tumor.Methods LEP,TNF-α,CEA,CRP and IL-6 levels in both pleural fluid and serum samples from 95 cases of pleural effusion (including 54 cases of malignant pleural effusion and 41 cases of tuberculous pleural effusion) were detected by immunoturbidimetry and enzyme linked immunosorbent assay (ELISA),respectively.The data with normal distribution and skewed distribution were analyzed by t test and rank sum test,respectively.Results In patients with tuberculosis and malignant tumor,significant difference was observed in serum LEP [(0.42±0.47) ng/ mL vs (1.80±0.91) ng/mL,t=9.666,P=0.0001],TNF-α [(30.88±24.72) pg/mL vs (59.83±30.93) pg/mL,t=4.917,P=0.0001],CEA [(0.22±0.30) ng/mL vs (5.67±3.60) ng/mL,t=ll.074,P=0.0001] and IL-6 [(146.48±54.90) pg/mL vs (20.51±11.62) pg mL,t=-14.449,P 0.0001] levels.Serum CRP levels of patients with tuberculosis and malignant tumor were comparable [(32.78±22.43) mg/mL vs (37.80±16.74) mg/mL,t =1.249,P=0.215].In pleural effusion of the two groups (tuberculous pleural effusion vs malignant pleural effusion),LEP [(0.69±0.65) ng mL vs (4.33±2.16) ng mL,t 11.711,P 0.0001],TNFα [(20.60±17.80) pg/ mL vs (40.40±20.60) pg/mL,t=4.926,P=0.0001],CEA [(0.10±0.17) ng/mL vs (4.02±2.49) ng/ mL,t=11.537,P=0.0001] and IL-6 [(87.80±48.40) pg/mL vs (9.30±5.50) pg/mL,t =-10.333,P=0.0001] levels were significantly different,while CRP levels [(27.34±17.93) mg mL vs (29.60± 13.40) mg mL,t =0.709,P =0.102] were comparable.Conclusion LEP,TNF-α,and CEA levels in both pleural effusion and serum samples from patients with malignant tumor are higher than those with tuberculosis,while IL 6 levels are quite opposite.Combined detection of these parameters can help the differential diagnosis of tuberculous and malignant pleural effusion.

Objective To explore the effect of transitional care model (TCM) on the prevention of complications in patients with severe cerebral infarction. Methods Sixty patients with severe cerebral infarction were randomly divided into the experiment group and the control group, 30 cases in each group. The control group was given conventional nursing and the observation group received TCM. The two groups were compared in terms of the incidence of complications and re-admission rate. Result The incidence of complications and the rate of re-admissions in the experiment group were significantly lower than those of the control group (P<0.05). Conclusion TCM can reduce the incidence of complications in patients with severe cerebral infarction, lower the incidence of hospital admission, relieve the pain of patients and improve the quality of life of patients.

Objective:To explore clinical significance of sinus heart rate turbulence (HRT) phenomenon in aged pa‐tients with stable angina pectoris (SAP) .Methods :A total of 120 aged SAP patients ,who received 24h DCG in our hospital from Jan 2013 to Oct 2015 ,were selected as SAP group .Meanwhile ,another 144 aged patients ,who re‐ceived 24h DCG examination simultaneously and coronary angiography results were normal ,were regarded as nor‐mal control group .According to coronary lesion severity ,SAP group was further divided into single vessel coronary disease group (single vessel group ,n=35) ,double‐vessel coronary disease group (double‐vessel group ,n=48) and multi‐vessel coronary disease group (multi‐vessel group ,n=37) .The 24h DCG ,HRT indexes ,including turbulence onset (TO) and turbulence slope (TS) ,were measured and compared among all groups .Results:Compared with normal control group ,there was significant rise in TO [(0.77 ± 0.37)% vs .(1.26 ± 0.92)% ] and significant reduc‐tion in TS [(5.45 ± 4.02) ms/RR interval vs .(1.53 ± 0.70) ms/RR interval] ,P<0.01 both ;significant rise in ab‐normal rates of TO (19.44% vs .42.50% ) ,TS (15.97% vs .31.67% ) and TO + TS (11.11% vs .30.83% ) in SAP group ,P<0.01 all .Compared with single vessel group ,there was significant rise in TO [(0.66 ± 0.22)% vs .(1.28 ± 1.11)% vs .(1.46 ± 1.20)% ] and significant reduction in TS [ (2.04 ± 0.82) ms/RR interval vs .(1.66 ± 0.38) ms/RR interval vs .(1.29 ± 0.58) ms/RR interval] in double‐vessel group and multi‐vessel group ,and TO of multi‐vessel group was significantly higher than that of double‐vessel group ,TS of multi‐vessel group was significantly low‐er than that of double‐vessel group , P<0.01 all .Conclusion:Sinus heart rate turbulence can be used as risk predic‐tor for aged patients with stable angina pectoris ,which can provide basis for clinical effective treatment and progno‐sis assessment .

[Objective] To investigate the mesenchymal stem cells (MSC) in treating acute lung injury (ALI) via ALI mouse model.[Methods] By monoclonal antibody Anti-GD2 of specific antigen ganglioside (GD2) only expressed on MSC as a carrier,new fluorescent molecule probe were synthesized through covalently coupling Anti-GD2 and fluorescent group CyDye mono-reactive NHS Esters (Cy7).Synthetic Anti-GD2-Cy7 and MSC were labeled by the specific binding of antigen and antibody in vitro.Total 84 balb/c male mice were selected and randomly selected 48 mice were divided into three groups:sham group (n =16),MSC+ ALI group (n =16),NS + ALI group (n =16).The lung histopathology and scores,lung W/D ratio and permeability of lung microvasculature were examined at 24 h,48 h after ALI mouse model.Other 36 mice were randomly divided into three groups:normal group (n =12),sham group(n =12),MSC +ALI group(n =12).Labeled MSC-GD2-Cy7 were transplanted into MSC+ALI group and sham group mice via tail vein injection.At 30 min,1 d,3 d,and 7 d post-MSC-GD2-Cy7 injection,the mice were sacrificed after anesthesia and then the lung was removed.Excised lung was detected on small animal fluorescent imager.[Results] Contrast to NS+ ALI group,the lung histopathology and scores,lung W/D ratio and permeability of lung microvasculature of MSC +ALI group were more greatly improved at both 24 h and 48 h.Fluorescent results showed that the signal intensity in thc lung of MSC +ALI group was significantly higher than that of sham group at each time point [(3.37 ± 0.02)× 10-4 vs (2.05 ± 0.04) × 10-4 scaled counts/s;(35.54 ± 0.47)× 10-4 vs (25.29 ± 1.48) × 10-4 scaled counts/s;(11.17 ± 0.75)×10-4 vs (6.09 ± 0.62)× 10-4 scaled counts/s;(3.10 ± 0.14) vs (0.00 ± 0.00)× 10-4 scaled counts/s;all P ＜ 0.05].[Conclusion] Our study showed that a proportion of cells migrated into normal and injured lungs 30 min after cell transplantation,and the cells started to recruit and largely gather in injured lungs at day 1 and persisted to day 7,these results suggest that MSC possess the ability to home into injured tissues.

AIM: To determine differentially expressed genes associated with cell adhesion and immune regulation in peripheral blood eosinophils from asthmatic patients. METHODS: Peripheral blood eosinophils were isolated from the asthmatic patients at the time of exacerbation and after improvement. Total RNA was extracted. Super SMART PCR cDNA was synthesized, suppression subtractive hybridization (SSH) and PCR-select differential screening technology were used to detect expressed genes. The differentially expressed genes were sequenced. RESULTS: High efficiency subtractive cDNA library was constructed successfully. Differential screening identified 15 differentially expressed genes, which were Charcot-Leyden crystal protein (CLC protein; galectin-10), putative pre-mRNA splicing regulator female-lethal (2D), aquaporin 9 (AQP9), IL-8, slingshot 2L (SSH-2L), PP1 catalytic subunit, beta isoform, helicase with zinc finger domain (HELZ), ?2-microglobin (?2-MG) and a gene associated with mitochondrion. CONCLUSION: Increased expression of these genes might be associated with eosinophil migration, adhesion and immune regulation. Intervention of these pathways may provide a theoretical base for future new targeting treatment for asthma.

Objective To explore the change of glyoxalase I in type 2 diabetic ocular muscles palsy (DOMP) and its associations with advanced oxidation protein products (AOPP) and oxidative stress. Methods 58 DOMP patients, 50 T2DM and 30 normal controls were enrolled in this study. Levels of blood lipids, fasting blood glucose, hemoglobin A1c, insulin, serum glyoxalase I, AOPP, malondialdehyde (MDA), superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) were measured. Homeostasis model assessment was performed to evaluate the status of insulin resistance (IR). Results Levels of high-density lipoprotein cholesterol, SOD and T-AOC were positively correlated with glyoxalase I and inversely associated to AOPP. Levels of triglycerides , low-density lipoprotein cholesterol , fasting blood glucose , hemoglobin A1c , IR and MDA were negatively correlated with glyoxalase I and positively related to AOPP. AOPP had an inverse association with glyoxalase I (r = -0.823, P < 0.001). Multivariate regression analysis showed that serum levels of glyoxalase I (Sβ = 0.554) and AOPP (Sβ= -0.469) were influencing factors of groups. Conclusion Serum glyoxalase I levels were significantly decreased in DOMP and correlated with AOPP and levels of oxidative stress , which suggest that glyoxalase I could play crucial roles on the development of DOMP.

Objective To investigate the effects of advanced oxidation protein products (AOPP) in type 2 diabetic ocular muscles palsy (DOMP). Methods 58 DOMP patients and 50 type 2 diabetes patients were included in the research. Hemoglobin A1c (HbA1c), blood glucose (FPG), fasting insulin (FINS) and triglycerides (TG), total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL) were measured and recorded. Homeostasis model assessment was performed to evaluate the status of insulin resistance (HOMA-IR), basal insulin secretion (HOMA-β) and the insulin sensitivity index (ISI). Serum AOPP was measured by enzyme linked immunosorbent assay. Unconditional logistic regression model was used to evaluate the influencing factors of DOMP. Results The DOMP group showed higher levels of plasma AOPP, TG, LDL, FPG, FINS, HbA1c and HOMA-IR, but lower levels of HDL, HOMA-β and ISI than those of the T2DM group. Unconditional logistic regression analysis revealed that AOPP was an independent risk factors for DOMP (OR =3.01, P = 0.002). Conclusion AOPP may be involved in the pathogenesis of DOMP. AOPP could be a useful indicator for monitoring the development of DOMP and for evaluating its severity.

Objective To investigate the effect of human umbilical cord mesenchymal stem cells (HUC-MSCs) on CD4+T cells in liver after hepatic ischemia-reperfusion injury (HIRI) in mice. Methods Two hundred and twenty-five mice were randomly divided into sham group, control group and MSC group, with 75 mice in each group. HIRI model mice were used in MSC group and control group. HUC-MSCs were injected in MSC group through inferior vena cava. Normal saline was injected in control group through inferior vena cava. Only laparotomy and abdominal closure were performed in sham group without blood vessel clipping. At 6, 12 and 24 h after operation, 15 mice of each group were randomly selected to sample eyeball blood and liver tissues, and the 30 mice left in each group were used to extract intrahepatic mononuclear cells. The number of intrahepatic mononuclear cells, percentage, number and positive rate of CD4+T cells in the mice of various groups at different time points were compared. The content of interleukin (IL)-17 in serum and liver tissue as well as expression levels of costimulatory molecules B7-1 and B7-2 messenger RNA (mRNA) in liver tissues of the mice at different time points were compared. Results At 12 and 24 h after operation, the number of intrahepatic mononuclear cells of control group was significantly higher than that of sham group, while the number of intrahepatic mononuclear cells of MSC group was significantly lower than that of control group (P<0.01-0.05). At 6, 12 and 24 h after operation, the percentage, number and positive rate of CD4+T cells of control group were significantly higher than those of sham group (all P<0.01), while the percentage of CD4+T cells of MSC group was significantly lower than that of control group (P<0.01-0.05). At 12 and 24 h after operation, the number and positive rate of CD4+T cells of MSC group were significantly lower than those of control group (P<0.01-0.05). At 6, 12 and 24 h after operation, the IL-17 contents in serum and liver tissues of control group were higher than those of sham group (all P<0.01), while the IL-17 contents in serum and liver tissues of MSC group were lower than those of control group (all P<0.01). At 6 h after operation, the mRNA expression level of B7-2 of control group was higher than that of sham group (P<0.05). At 12 and 24 h after operation, the mRNA expression levels of B7-1 and B7-2 of control group were higher than those of sham group (all P<0.01), while the mRNA expression levels of B7-1 and B7-2 of MSC group were lower than those of control group (all P<0.01). Conclusions HUC-MSCs inhibits the number of CD4+T cells and the secretion of IL-17 in liver after HIRI, as well as decreases the number of intrahepatic mononuclear cells and the mRNA expression of B7-1 and B7-2, thereby alleviating HIRI.

OBJECTIVETo investigate the effect of 1,25-dihydroxyvitamin D3 (1,25VD3) on house dust mites (HDM)-induced expression of thymic stromal lymphopoietin (TSLP) in human airway epithelial cells in vitro.

METHODSHuman airway epithelial 16HBE cells were incubated with 200, 400, and 800 U/L in the absence or presence of 1,25VD3 (10(-8) mol/L) for 6 h and 24 h, and TSLP mRNA and protein expressions in the cells were assessed using quantitative PCR and ELISA.

RESULTS16HBE cells incubated with HDM at 200, 400, and 800 U/L showed significantly increased TSLP mRNA and protein expressions (P<0.05). Pretreatment of the cells with 1,25VD3 obviously lowered 400 U/L HDM-induced TSLP expressions (P<0.05), but 1,25VD3 added along with HDM in the cells did not produce significant effects on TSLP expressions (P=0.58).

CONCLUSIONBoth 1,25VD3 and HDM can induce TSLP expression and release in 16HBE cells, but pretreatment with 1,25VD3 can decrease HDM-augmented TSLP expression in the cells.

Animals ; Bronchi ; cytology ; Calcitriol ; pharmacology ; Cell Line ; Cytokines ; metabolism ; Epithelial Cells ; drug effects ; metabolism ; Humans ; Pyroglyphidae